The search for effective treatments for liver disease remains a challenging medical issue. There are many causes of liver failure including anatomical defects leading to progressive liver disease, drug induced liver injury, metabolic liver disease, hepatic neoplasms, vascular injury affecting the liver and viral hepatitis. Known hepatitis vaccines are not always available and hepatitis C, for instance, kills an estimated 8,000-10,000 people in the United States per year. Without effective intervention that number is predicted to triple in the next 10-20 years. National Institutes of Health, Consensus Development Statement, "Management Of Hepatitis C" Mar. 24-26, 1997.
Although the liver has tremendous capacity to regenerate itself, liver damage can inhibit or abolish this regenerative capacity. There is currently no machine which is able to replace liver function. Currently, the only effective treatment for liver failure is to perform a liver transplant.
In 1994, only 10% (approximately 3,652 people) of those awaiting donor livers received a transplant. (United Network for Organ Sharing, UNOS). In addition, there are many complications associated with liver transplants. Even after matching donor and recipient, rejection of the graft organ often occurs. Immunosuppressants given to reduce the chance of graft rejection cause their own set of problems. Graft versus Host Disease (GVHD), a harmful immune system effect where lymphocytes within tissue that was grafted from a different individual attacks the tissue in its new location can also occur.
Due to the severe shortage of donor livers and complications of associated with transplants, an alternative to human liver transplantation would save many patients from suffering and premature death associated with acute liver failure. Health expenditures would also be reduced, as liver transplants are currently performed at an average cost of approximately $150,000 per transplant. PROGRESS (1997), American Liver Foundation. An alternative to liver transplantation that avoids host immune rejection would therefore be particularly useful.
A great deal of attention has been focused on the possibility of treating liver failure with regenerative hepatocytes. Grompe et al. (1997), Amer. J. Pathol., in press, have shown that a single mouse hepatocyte can expand through at least 69 cell divisions, generating 7.times.10.sup.20 cells. Since an average mouse liver contains approximately 3.times.10.sup.7 hepatocytes, one hepatocyte has the capacity to generate a number of cells equivalent to 10.sup.13 mouse livers. However, relative to other regenerative tissues, liver regeneration is a complex response involving proliferation of all the existing mature cells of the liver, rather than on a small group of progenitor cells. Michaelopolous et al. (1997) Science 276:60-65. Liver stem cells have not been identified microscopically and it is postulated that they may not exist due to the long life span of hepatocytes as well as differentiated hepatocytes' ability to regenerate in response to liver cell loss. Aterman et al. (1992) J. Cancer Res. Clin. Oncol. 118:87-115; Potten et al. (1990) Development 110:1001-1020.
In order to test liver regenerative capability, several animal models have been generated. A transgenic mouse bearing the urokinase-type plasminogen activator (uPA) coding sequence fused to the albumin (Alb) enhancer/promoter causes neonatal hemorrhaging in mice hepatocytes. Heckel et al. (1990) Cell 62:447-456. While this mouse model has been used in the study of the regenerative potential of transplanted hepatocytes (Sandgren et al. (1991) Cell 66:245-256; Rhim et al. (1994) Science 263:1149-1152; WO 94/02601), there are disadvantages in using this mouse to show that transplanted cells can restore liver function. In particular, the toxic uPA transgene is deactivated by DNA rearrangement in isolated hepatocytes which in turn leads to repopulation of the entire liver by cells that do not express uPA. As a result of this loss of the transgene, only one half of the transgenic mice die at birth, while the other half survives and expresses normal plasma uPA concentrations within 2 months of birth. Sandgren et al. (1991), supra.
A mouse model of a human liver disease has recently been developed by the present inventor which is useful for examining the restoration of liver function. Hereditary Tyrosinemia type I (HT1) is a metabolic disease caused by a lack of the enzyme fumarylacetate hydrolase (FAH) and is characterized by severe liver dysfunction in childhood, renal tubular damage, and hepatocellular cancer. The development of the FAH-mutant mouse model (Grompe et al. (1993) Genes & Dev. 7:2298-2307) is especially useful because therapy for HT is usually human liver transplantation. Treatment with the drug NTBC (2-(2-nitro-4-triflouro-methylbenzoyl)-1,3-cyclohexedione) prevents the neonatal lethality and liver dysfunction in the transgenic mouse. When NTBC is withdrawn, the FAH-mutant mice develop a liver dysfunction with a similar phenotype to humans suffering from HT. Grompe et al. (1995) Nature Genet. 10:453-460.
The FAH deficient mouse model has several advantages over the urokinase model described above. First, the viability of the urokinase transgenics is low and surgical procedures are difficult because of the systemic bleeding induced by this secreted protein. In contrast, NTBC treated FAH mutant mice are healthy and viable. Second, there is a high rate of spontaneous reversion (loss of the transgene) in the urokinase transgenics, so that the liver of these animals will have self-corrected by the time the animals are 1 month old. (Rhim et al. (1994) Science 263:1149-1152; Sandgren et al. (1991) Cell 66:245-256). For this reason, transplantation of wild-type cells into urokinase mice must take place very early in life, usually at less than 15 days in order for selection to take place. This makes surgical manipulations such as intraportal injections difficult. In contrast, FAH deficient animals can be kept on NTBC as long as desired and then transplanted as adults. The mutation in the FAH mice (FAH.DELTA..sup.exon5) does not spontaneously revert. Accurate quantification of nodules is also a problem in the urokinase transgenic model because the spontaneous reversions make it difficult to distinguish nodules arising from transplanted cells from nodules arising from reversion.
A third advantage of the FAH system is that the selective pressure in FAH mutant mice can be turned off or on using either NTBC or homogentisic acid. This permits flexibility in the selection process, an important consideration for successful xenograft experiments. If, for example, the transplanted cells take time to repopulate, a low level or intermittent NTBC treatment will allow the recipient animals to survive while selection is occurring. Fourth, the selection in FAH deficiency is a positive selection for an added gene. FAH deficient cells can be transduced and marked with an FAH expressing construct, such as retroviruses. The ability to tag cells is invaluable in lineage experiments. In contrast, selection in the urokinase system is a negative selection for the loss or absence of the transgene and selection for retroviral marking is impossible.
A further advantage of the FAH mouse model is that HTI, with its high .alpha.-fetoprotein level and continuous liver regeneration, represents exactly the kind of pathological condition in which one would expect facultative liver cells to be activated. The autocrine and paracrine growth environment in these livers is likely to the meet the requirements for expansion and growth of progenitor cells, including from xenogenic sources.
Using the FAH-mutant model, researchers have shown that wild-type hepatocytes have a selective advantage over FAH deficient cells and can repopulate the liver when mice are removed from NTBC. Overturf et al. (1996) Nature Genet. 12:266-273 This model has also been used to show that hepatocytes can be corrected by gene therapy with a recombinant adenovirus expressing FAH. Overturfet al. (1997) Human Gene Ther. 8:513-521. In addition, Overturf et al. (1996), Nature Genet. 12:266 report that as few as 1000 hepatocytes can restore liver structure and function in a mouse liver. More than 90% of the resulting hepatocytes were found to be FAH positive.
However, the applicability of these hepatocyte studies is limited as most patients with liver failure do not have enough unaffected hepatocytes to regenerate a healthy, functioning liver. As described above, transplantation of heterologous liver cells involves significant risk associated with the host's immune rejection of the transplanted cells. Therefore, it would be useful to have an autologous source of unaffected cells capable of repopulating the liver.
Scarpelli et al. (1981) PNAS 78:2577 describe how acinar pancreas cells treated with the carcinogen N-nitroso-bis(2-oxopropyl)amine (BOP) appear similar to hepatocytes. Similarly, Lalwani et al. (1981) Carcinogenesis 2:645 describe how hepatocyte-like cells can be induced in the pancreas of rats by feeding the animals a diet containing (4-chloro-6-(2,3-xylidino)-2pyrimidinylthio) acetic acid, a peroxisome proliferator. Reddy et al. (1991) Digestive Dis. Sci. 36(4):502 describe methods of inducing hepatocyte-like cells in rat pancreas by maintaining the rats on a copper-deficient diet containing the copper chelator, triethylenetetramine tetrahydrochloride (trien). Under these conditions, the pancreatic acinar cells are depleted and hepatocyte-like cells appear. Rao et al. (1989) Am. J. Pathol. 134(5):1069-1086 report that after 8 weeks on the copper-deficient diet 60% of the volume of the pancreas was occupied by hepatocyte-like cells. These studies do not report on the function of either the pancreas or the liver.
Dabeva et al. (1995) Am J. Pathol. 147:1633-1648 describe how pancreatic epithelial cells transplanted into the livers of rats proliferate and express liver specific genes. Chen et al. (1995) Am J. Pathol. 147(3):707-717 describe how rat pancreatic epithelial cells genetically labeled with .beta.-galactosidase resemble hepatocytes 4 to 8 weeks after transplantation into rat livers. Pancreatic epithelial progenitor cells, identified by their expression of dipeptidyl peptidase IV (DPPIV), were transplanted into the livers of DPPIV-mutant rats and found by histochemical analysis to show characteristics of mature hepatocytes and physical continuity with endogenous hepatocytes within the liver plates. Sigal et al. (1995) Cell. Mol. Biol. Res. 41(1):39-47; Dabeva et al. (1996) J. Invest. Med. 44(3):206A.
WO 96/40872 describes methods for isolation and in vitro expansion of pancreatic progenitor cells isolated from the bile duct. In this system, the pancreas cell population is contacted with a proliferative agent such as a growth factor that specifically stimulates growth of certain distinct groups of pancreatic progenitor cells. Progenitor cells within the culture are identified by changes in cell proliferation and morphology. The pancreatic progenitors are then isolated from other pancreatic cells using techniques based on specific cellular markers. Progenitor cells which differentiate into pancreatic islet cells or hepatocytes and are identified by the expression of various specific cellular markers can be further cultured under conditions allowing differentiation into various cell lineages such as hepatic and pancreatic. This method does not assay the ability of cells to regenerate a functioning liver because the pancreatic progenitor cells and hepatocytes are characterized only by their physical appearance and cellular markers. Thus, these studies do not provide evidence that transplanted cells have the ability to restore liver function.